Effect of Saponin Extracted from Passiflora alata Dryander (Passifloraceae) on development of the Spodopterafrugiperda (J.E. Smith) (Lepidoptera, Noctuidae)

Thesaponins are glycosides with wide distribution among plants which may be toxic for herbivorous arthropods. Their insecticidal activ ity may be associated to the ability of producing alterat ions in the feeding behavior, in the molting process and causing death. This study evaluated the lethal and sub lethal effects of a saponins extract, obtained from Passifloraalata on Spodopterafrugiperda, by ingestion tests with artificial diet. Nine solutions with increasingextract concentration and a control solution with sterile distilled water were used in the test. In total 1500 insects were used in 5 repetitions, 30 replicates per concentration. Considering the total mortality, all treatments differed statistically from the control one, but not among them. Most of the observed effects were sub lethal, in which 68.3% insects presented deformation. The number of deformed insects per treatment increased as the extract o f saponins concentration increased. In conclusion the mortality revealed significant difference between control and other treatments, which indicated the potential of saponins present in P. alata as a control agent of S. frugiperda. This potential needs to be better evaluated, especially if the facilities of raising and large scale production of the plant are considered.


Introduction
Th e res earch es d es ign ed at id en t ify in g n ew to xic substances in o rder to con tro l ag ricu ltu ral p lagu es are const ant in th e act iv it ies relat ed to man ag emen t an d integrated control of plagues nowadays. This is partially a resu lto f the kno wledg e o f the negativ e imp acto f th e chemical p roducts used in the ecosystem as well as the possibility of evolution of the resistance of the target insects to the products used, whether these are chemical and/or biological. A great number of these researches aredirected to the evaluat ion o f to xicity of secondary metabo lite of p lan ts , s u ch as p h en o ls , alkalo id s , g lu co s in o lates , cyan ogen ic g lycos id es and sapon ins . 1 M ore th an 2000 species of plants are kno wn to possess some insecticidal activity. In many cases plants have a history as traditionalmedicines or are used to kill or repel insects. In spite of the various studies available about the entomotoxic effects of extracts of different species of plants, their application is still incip ient. 2 The saponins are glycosides with detergent properties, due to the presence of hydrophobic (aglycones) and hydrophilic (sugar) co mponents with wide distribution among plants which may be to xic forherb ivorous arthropods such as mites, beetles and Lepidoptera, among others. [3][4][5][6][7] Their insecticidal activ ity may be associated to the ability of producing alterations in the feeding behaviour, in the molt ing process, of interacting with hormones that regulate the growth and causing death in the different stages of development. [8][9][10][11] Their wide spectrum of action reaches a great number of herbivores and its amplitude of physiological impacts makes these substances an excellent model for the study of the effect of natural substances with insecticidal activ ities. 8 The passion fruit,Passifloraalata, is one of the main species of economic importance of the gender cultivated in Brazil, wh ich is used mainly as fruit and for juice and sweets production as well as a med icinal plant. [12][13][14][15][16] Thesaponins are the main substances of its secondary metabolism. Five types of saponins were isolated and identified in its leaves: one is a steroid and four are triterpenic (3)(4)(5)(6).

17-18
The fall armyworm Spodopterafrugiperda (J. E. Smith) (Noctuidae) is a polyphagous insect, using more than 50 species of plants as food supplies, distributed in over 20 families, 19 predominantly in g rasses, among them corn and rice, which are important cultures for Brazilian and world economy. In rice,S. frugiperda is considered a pestof the initial phase, since it attacks the seedlings in the beginning of their develop ment, feeding on the leaves and cutting the new stalk close to the ground, 20 causing losses of 14 to 24% of the grains. Depending on the population level, the destruction of the crop can be total. 21 In maize, the attack can occur fro m the seedling stage until the tasseling and the formation of spikes. In the later attacks specimens between the stalk and the cob can be found, where they penetrate the female inflorescence destroying the grain. 22 The effect of saponins extract of some plants on the development of the Spodopteraspecieswas demonstrated in studies with S. litura. 2,4,[23][24][25][26][27] The results of these studies indicate alterations in the feeding behaviour, lengthening of the larvae and/or pupa stages, increased mortality and reduced fertility.
The study of plants with potential insecticidal act ivity is important to discover new active molecules, which may be isolated fro m p lants or synthesized, or a molecu le-prototype for structural changes to obtain a more act ive compound.
This work evaluated the effects of saponins extracts of PassifloraalataonSpodopterafrugiperda, identifying the sub lethal and lethal effects of these substances in different development stages of this species.

Preparati on and Characterizati on of the Saponin Extracts
To obtain saponin ext ract the dried leaves were submitted to macerat ion with 70% ethanol. The ethanol was filtered and placed in a rotary evaporator; the aqueous residue was extracted successively with chlo roform, ethyl acetate and nbutanol. The evaporation of n-butanol fraction resulted in a fraction consisting main ly of saponins, which will be called henceforth saponin extract. 17 Thesaponin extract is characterized as a yellow-orange powder.

Obtenti on and Rearing of Insects
The establishment of S. frugiperda began with immaturelarvae co llected in rice plantations, with the collaboration of researchers at the Estação Experimental do Arroz (EEA) of the InstitutoRiograndense do Arroz (IRGA), located in Cachoeirinha, RS. The insects were reared in the room for the creation of insects of the Laboratory of Microbiology of the Un iversity of Vale do Rio dos Sinos (UNISINOS), heated to 26°C, 75% RH and 12h photo phase. The adults were kept in plastic cages, fed with a glucose solution of 10%. Eggs were collected three times a week and put in gerbo x with d iet. Five or six days after hatching, the larvae were individualized in plastic cups, and maintained with art ificial Po itoutdiet, 28 where they remained until they became pupae. As a routine procedure adopted, the pupae were identified, separated by sex, kept in sleeves with moistened filter paper and covered with tulle, until emergence of adults.

Bioassay
For the test with S. frugiperda nine solutions with increasing concentrations of saponin ext ract were used due to the 2x (312, 625, 1250, 2500, 5000, 10000, 20000, 40000 and 80000pp m) and a control of sterile distilled water. A solution of 20ml was prepared for each concentration, which, after use, was stored at 4°C. In this case, the extracts were diluted in sterile d istilled water. The second instar larvae of S. frugiperda were maintained for seven days in mini acry lic plates (35mm diameter) containing Po itout diet, where 100μL of the solutions were applied on the diet. After thisthey were transferred to plastic cups containing only artificial diet, where they were observed until emergence of adults for the evaluation of possible sub lethal effects ( Figure 1). Damages were evaluated under stereoscopic microscope and classified according to intensity (low, mediu m and high) ( Figure 2).

Data Analysis
Data were tested for normality using the chi-squared and Shapiro-Wilks and the homogeneity with the tests of Hartley and Bart lett. When these criteria were satisfied, they were compared by an ANOVA followed by mult iple tests of Tukey. Otherwise, they were co mpared by Kruskal-Wallis follo wed by Dunn's mult iple test. Statistical analysis was performed with the aid of TOXTAT Soft ware version 3.3. 29 To compare mo rtality rates between larvae and pupae of S. frugiperda the Student t test was conducted for different means and to analyse the deformation of the same species a simp le linear regression was made. 30    T1  T2  T3  T4  T5  T6  T7  T8  T9  T1  T2  T3  T4  T5  T6  T7  T8  T9   Larvae  1  1  3  4  9  7  8  8  10  0  0  0  0  0  0  0  0  0   Pupae  2  5  4  4  6  3  4  6  1  1  1  0  0  2  6  9  2  1   Adults  20  32  32  25  36  24  30  29  42  0  0  1  0  1  10  2  2  3   Total  23  38  39  33  51  34  42  43  53  1  1  1  0  3  16  11  4  4 The quantitative and qualitative results of the experiments are indicated on tables 1 and 2, respectively Considering the total mo rtality (larvae and pupae) all treatments differed statistically fro m control but not among them ( Figure 3) (p<0.05). Co mparing the mortality of larvae with the mortality of pupae in each treatment, it was observed that the rate of larval mortality was higher than that of the pupae from the control concentration until the concentration 2500pp m. At higher concentrations (5000 to 80000 pp m), these rates showed no significant difference ( Figure 4). There were 441 o f larvae and 268 of pupae deaths, i.e. 8.1% and 29.4% of o rganisms exposed to saponin, respectively, totalizing 17.9% of the insects used in bioassays.

Results
Of the 441 larvaethat died during the tests, 121 (27%) died with some deformity, of which 45 (37.2%) died during the process ofmolt, unable to release in who le or in part of mo lting, 8 (6.6%) had points of necrosis and 5 (4.1%) some parts of the body distended Of the 268 dead pupae, 107 (39.9%) died with some deformity, of which 27 (25.2%) had retention of morphological characters of larvae, 88 (82.2%) d ied during the emergence of the adult failing to release in whole or in part, 5 (4.7%) had deformit ies in the form of globular evaginations of the cuticle, 23 (21.5%) had points of necrosis in the cuticle and 11 (10.3%) sclerotinizat ion failures, leaving the internal organs exposed. Some specimens had more than one type of deformation, such as evaginations and points of necrosis (2.93%), or points of necrosis around the failu res of the cuticle (0.97%). Often, when reviewing the experiments, the pupae with failure sclerotinization were found still alive, being dead in 70% alcohol immediately to avoid the suffering of the indiv idual. The main deformation observed in 599 adults obtained after the bioassays was in the reg ion of the wings, where 200 (33.4%) insects showed little or no distension of the wings.
A total of 1025 insects (larvae, pupae and adults) were observed withdeformations (76% of organis ms) ( Figure 5). These organisms were screened according to the intensity of their deformat ions: 612 (59.7 %) had deformities of low intensity, 144 (14.05%) of average intensity and 269 (26.2%) of high intensity (Table 3) The linear reg ression obtained from the number of deformit ies observed in insects exposed to the saponin extracts, was a straight up line, wh ich shows that there is a tendency to increase the number of deformed insects as the concentration of extract saponins increases (Figure 6)..     In summary the deformit ies were observed in 76% of organisms exposed to saponin. These deformations were lin ked, primarily, to the process of mo lt, as 45 larvae of S. frugiperda died during mo lting process, failing to release in whole or in part of molted cuticle. Besides the problems related to the process of molt, the organis ms that had points of necrosis in the larvae stage were located mainly in the thorax and the pupae stage and adults, in most cases, in the region of the wings. Moreover, problems were observed in sclerotinization of pupae, which opened up large holes in the region of the wings, leaving the interior of organis ms exposed. In organisms of control treatment no deformities were observed.

Discussion and Conclusions
This study identified the impact o f saponins extract on the different stages of development of S.frugiperda.Besides the mortality of larvae and pupa, the presence of structural deformit ies was observed in all stages. When considered the total impact of the treatments 47.4%of the adults emerged without structural problems identified by optical microscopy. The effects of the saponin extract on S. frugiperdamay be associated to a deterrent toxic act ion and/or by its interactions with substances responsible for the different stages of development of the specie.
The deterrent effect reduces the consumption of food producing nutritive deficiency causing deficient growth or deformit ies, wh ich may lead to death or inhibit the progress for the next stage. 26,27,31,32 Among the alterations observed, the presence of necrosis points stood out, sclerotinizat ion problems and retention of larvae characters and tumors.These results are similar to the ones registered, 21 for the biotypes of corn and rice of S. frugiperdausing different extracts of different species of plants.It is important to highlight that alterations in the length of the larvae and pupal stages were not observedin this study, as registered for S.littoralis 23 e for S. frugiperda. 33 The differences in these results may be associated to the variation in the composition of saponins among the plant species used for the acquisition of the extracts. 3,34 The physiological basis of the to xicity of saponins has not been fully elucidated, but we know that there is a great interaction between them and the cell membranes, with effects on the hydrophobic-lipophilic balance and permeab ility of these because they are capable of forming complexes such as sterols, for instance, cholesterol. 10,11,35 The toxicity of saponins on arthropods may derive fro m their ability of interacting with free steroids of the intestine and/or of inhibit ing the digestive proteases, reducing the rates of digestion and absortion. 8,35,36 The mortality and deterrence caused by saponin were also observed in experiments that evaluated the effects of different types of saponin for aphids, 38,39,42 nematodes, [40][41][42] beetles 31,43,44 and Spodoptera sp. 2,5,6,25,27 A fact that drew attention was the appearance of specimensin the intermediary period between larvae and pupae (1.8% o f exposed organisms). Apparently, the organisms began the process of becoming pupae and could not complete it, which led to death. The appearance of intermediate indiv iduals between pre-pupae and pupae can occur when the activity of juvenile hormone, which controls the metamorphosis, is affected. 32 Furthermore, the metamorphosis may have been affected because of saponins form co mplexes with sterols, like cholesterol, both in the intestine in cell membranes of insects. These complexes formed by saponin make sterols unavailable, influencing the synthesis of ecdison, one of the hormones involved in the process of molt. It has been shown that insects are unable to produce these sterols, removing them entirely fro m the diet. 10,11,34,36,39 The statements of these authors may account for the large number of deaths of larvae and pupae during the processes of molt and emergence of pupae malformed, beyond the failures of sclerotinizat ion observed.
In conclusion the mortality of S. frugiperda showed significant difference between control and other treatments. It was observed that the death of the specimens was not immed iate after the ingestion of saponin, but slow and gradual, which apparently occurred under the influence of saponin in the metabolism of the larvae, pupae and adults. These results indicate the potential of saponins present in P. alataas a control agent of S.frugiperda. This potential needs to be better evaluated, especially if the facility of raising and large scale production of the plant is considered.